Comparative effects of bone morphogenetic proteins and Sox9 overexpression on matrix accumulation by bovine anulus fibrosus cells: implications for anular repair.

STUDY DESIGN: An in vitro biologic study comparing the effects of a series of bone morphogenetic proteins (BMPs) and Sox9 on the extracellular matrix accumulation by bovine anulus fibrosus (AF) cells. OBJECTIVE: To compare the effects of adenoviral-mediated overexpression of various BMPs and Sox9 on extracellular matrix accumulation by AF cells in vitro. SUMMARY OF BACKGROUND DATA: Repair of the disrupted AF, which is perceived to be a potential therapy to diminish nucleus pulposus (NP) herniation, may also offer a treatment strategy for severe symptomatic degenerative disc disease. To date, no systematic comparison of a large group of growth factors in the AF has been published. In this study, we compared the effects of the adenoviral-mediated overexpression of 12 BMPs and Sox9 on extracellular matrix production by AF cells. METHODS: Adult monolayer-cultured bovine AF cells were transduced with adenoviral vectors containing human BMP and green fluorescence protein (GFP) genes (AdBMPs), or Sox9 and GFP genes (AdSox9), or GFP gene alone (AdGFP, as negative control). Proteoglycan and collagen accumulation, and cell proliferation were measured for each of the treatment groups 6 days after viral transduction. RESULTS: AF cells transduced with BMP-2, -3, -5, -7, -8, -12, -13, -14, and -15, and Sox9 accumulated significantly more collagen than AF cells transduced with AdGFP (control). AF cells transduced with AdBMP-2, -4, -7, -10, -12, and -13, and AdSox9 accumulated significantly more proteoglycans than AF cells transduced with AdGFP. CONCLUSION: We have demonstrated the relative effectiveness of 12 different BMPs and Sox9 on the stimulation of proteoglycan and/or collagen accumulation by AF cells. This study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by AF. This information should prove useful to those seeking to develop a strategy for repair of the AF in humans.

Transduced bovine articular chondrocytes affect the metabolism of cocultured nucleus pulposus cells in vitro: implications for chondrocyte transplantation into the intervertebral disc.

STUDY DESIGN: Biologic study on the effects of coculture of bovine articular chondrocytes transduced ex vivo with genes expressing bone morphogenetic proteins (BMPs) on nucleus pulposus (NP) cells. OBJECTIVE: To evaluate the effects of bovine articular chondrocytes transduced with adenoviruses expressing various BMPs on proteoglycan and collagen production, and cellular proliferation of NP cells in vitro. SUMMARY OF BACKGROUND DATA: Matrix synthesis by intervertebral disc cells is promoted by exposing the cells to growth factors or delivering genes that permit sustained expression of growth factors. We propose a novel therapeutic approach involving delivery of autologous chondrocytes, transduced ex vivo with bioactive proteins, to provide both the cells and proteins required to stimulate disc healing. METHODS: Adult bovine articular chondrocytes were transduced with adenoviruses (Ads) expressing either BMP-2, 4, 5, 7, 10, or 13 and plated as monolayers. Bovine NP cells encapsulated in alginate beads were cocultured, floating in the medium. Proteoglycan and collagen accumulation, and NP cell proliferation were measured after 6 days of coculture. As a positive control, beads were cocultured with articular chondrocytes in the presence of rhBMP-7. RESULTS: NP cells cocultered with articular chondrocytes transduced with BMPs-2, 4, 7, and 10 accumulated significantly (P < 0.05) more proteoglycan than when cocultured with chondrocytes transduced with AdGFP (control) [AdBMP-2: 23.6%; AdBMP-4: 27.0%; AdBMP-7: 129.1%; AdBMP-10: 102.1% increases respectively]. Collagen accumulation was significantly (P < 0.05) increased by NP cells cocultured with articular chondrocytes transduced with BMPs-2, 4, 5, and 7. [AdBMP-2: 104.6%; AdBMP-4: 40.6%; AdBMP-5: 58.6%; AdBMP-7: 55.5% increases respectively]. NP cells proliferated when cocultured with articular chondrocytes transduced with AdBMP-2 and -7. CONCLUSIONS: Bovine NP cells are stimulated to produce proteoglycans and collagen when exposed to chondrocytes transduced with genes for various BMPs. If applied to the treatment of disc degeneration, this strategy could provide the disc with not only metabolically active chondrocytes but also promote matrix replenishment by stimulating native NP cells.

Age-related differences in the accumulation and size of hyaluronan in alginate culture.

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.